Microfluidic chips and assay systems

ABSTRACT

The systems and methods described herein include a microfluidic chip having a plurality of microfeatures interconnected to provide a configurable fluid transport system for processing at least one reagent. Inserts are provided to removably interfit into one or more of the microfeatures of the chip, wherein the inserts include sites for interactions with the reagent. As will be seen from the following description, the microfluidic chip and the inserts provide an efficient and accurate approach for conducting parallel assays.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to Provisional Application No.60/760,552, filed on Jan. 19, 2006, and incorporated herein by referencein its entirety.

FIELD OF THE INVENTION

The systems and methods described herein generally pertain to the fieldof microfluidics. In particular, these systems and methods pertain tomicrofluidic diaphragm structures, microfluidic chips, portableautomated microfluidic reagent processing systems, and fabrication anduse thereof.

BACKGROUND AND OBJECTS OF THE INVENTION

“Microfluidics” generally refers to systems, devices, and methods forprocessing small volumes of fluids. Because microfluidic systems canintegrate a wide variety of operations to manipulating fluids, such aschemical or biological samples, these systems have many applicationareas, such as biological assays (for, e.g., medical diagnoses and drugdelivery), biochemical sensors, or life science research in general.

One type of microfluidic device is a microfluidic chip. Microfluidicchips may include micro-scale features (or “microfeatures”), such aschannels, valves, pumps, and/or reservoirs for storing fluids, forrouting fluids to and from various locations on the chip, and/or forreacting fluidic reagents.

However, existing microfluidic systems lack adequate mechanisms forallowing controlled manipulation of multiple fluids except viaprescribed flow patterns, hence limiting the practicality with which thesystems can be utilized in various chemical or biological assays. Thisis because real-world assays often require repetitive manipulation ofdifferent reagents under continuously varying conditions.

Moreover, many existing microfluidic devices are restricted for onespecific use and cannot be easily adapted or customized for otherapplications without being completely redesigned. These devices lackmodularity, and therefore cannot share common device components thatallow one design to perform multiple functions. This lack of flexibilityleads to increased production costs as each use requires the productionof a different system.

Furthermore, many existing microfluidic systems lack any means forstraightforward end-point assays that are able to easily detectinteractions or existence of analysts resulting from the assays. By wayof example, visual detection of sample color changes after an assay isoften used to evaluate the assay results, but this technique is rarelyapplied in a microfluidic system.

Thus, there exists a need for improved microfluidic systems forprocessing fluids for analysis of biological or chemical samples. It isdesired that the systems are mass producible, inexpensive, andpreferably disposable. It is desired that the systems be simple tooperate and that many or substantially all of the fluid processing stepsbe automated. It is desired that the systems be customizable, and bemodular such that the system can be easily and rapidly reconfigured tosuit various applications. It is desired that the systems be able toprovide straightforward and meaningful assay results.

SUMMARY

The system and methods described herein, in one embodiment, include aplastic microfluidic chip configured to process one or more reagents.The chip may comprise various microfluidic features including valves,pumps, channels and reservoirs. The micro-features are interconnected toallow various combinations of fluid flow patterns that can be userspecified and tailored to a particular application. In particular, thechip allows for the transport of one or more reagents from respectivereagent reservoirs on a reagent cartridge to multiple assay channels viaa transport structure. The transport is directed by the automatedoperation of pneumatically driven pumps and valves. By coordinating theflow of reagent from the reagent reservoirs to the channels bothspatially and temporally using the automated methods described herein, auser can efficiently perform biological immunoassays.

In one aspect, the microfluidic chip includes a plastic substrate havinga plurality of channels, a distribution structure for introducing areagent into at least one of the channels, and a configurable transportsystem for controllably directing a flow of the reagent in the channels.

In one aspect, the channels include a plurality of inlet channels, aplurality of outlet channels and a plurality of assay channels. Theconfigurable transport system comprises a distribution valve connectedto the inlet channels and outlet channels for distributing reagents tothe assay channels. The assay channels are configured for conductingbiological assays.

In one aspect, the inlet channels, outlet channels, assay channels anddistribution structure are disposed in the substrate body.

In one aspect, the porting device is a separate reagent cartridge thatis detachably coupled to a top surface of the substrate and has aplurality of reagent reservoirs fluidly communicating with therespective inlet channels. The inlet channels are individually valvecontrolled to deliver reagents from the respective reagent reservoirs tothe assay channels through the distribution valve and the outletchannels.

In another aspect, there is a buffer reservoir aligned with an inletchannel to the distribution valve. The buffer reservoir features asubstantially larger storage volume than the individual reagentreservoirs for storing a washing buffer. A diaphragm valve locatedbeneath the buffer reservoir controllably releases the washing bufferinto the assay channels through the distribution valve.

In another aspect, the invention includes one or more shuttle reservoirsand outlet reservoirs for storing reagents and buffer that aretransported during reaction incubation. The shuttle reservoirs areconnected to the corresponding outlet reservoirs through respectiveassay channels. The volumes of a shuttle reservoir and an outletreservoir are substantially larger than the volume of an assay channelso that a reaction reagent in the assay channel can be transported intothe shuttle reservoir and/or the outlet reservoir during reactionincubation.

In another aspect, the invention includes an on-chip waste reservoiraligned with an outlet channel to the distribution valve. The wastereservoir features a substantially larger storage volume than the bufferreservoir for storing all used reagents and washing buffer. Anindependently actuated diaphragm valve located beneath the wastereservoir regulates fluid flow into the waste reservoir from the shuttleand/or outlet reservoirs via the distribution valve.

In another aspect, the invention includes one or more bi-directionalfluidic pumps each coupled to at least three valves respectivelycontrolling a fluid flow through an assay channel, a shuttle reservoirand an outlet channel to the distribution valve. The pump-and-valvesstructure enables multiple fluid drawing and delivery patterns such asfrom a reagent reservoir to a shuttle reservoir, from a reagentreservoir to an assay channel to an outlet reservoir, from a shuttlereservoir to an outlet reservoir via an assay channel, from an outletreservoir to a shuttle reservoir via an assay channel, from an outletreservoir to a waste reservoir and from a shuttle reservoir to a wastereservoir.

In another aspect, the porting device comprises a separate reagent chipincluding the inlet channels, the distribution valve and a plurality ofreagent reservoirs. The reagent reservoirs are aligned with the inletchannels for introducing reagents to the distribution valve. The portingdevice also includes a ducting chip having the outlet channels disposedtherein. The ducting chip is adapted to detachably couple to the reagentchip and the substrate for introducing the reagents from the reagentchip to the assay channels in the substrate. The separation of anapplication chip into several modules allows greater design andfabrication flexibility, the utilization of a variety of chip materialsand the repetitive usage of the reagent cartridge.

In another aspect, the invention includes an insert disposed in a voidvolume of an assay channel for conducting biological assays or chemicalreactions, wherein the assay channel is configured to receive the insertand prevent a reaction surface of the insert from contacting the channelsurface.

In another aspect, the assay channel is adapted to receive the insertfrom an opening of the outlet reservoir connected to the assay channel.

In another aspect, the void volume of the assay channel includes anopening to the top surface of the substrate wherein the insert can bedisposed, and a lid for removably covering the opening of the voidvolume.

In another aspect, the reaction surface of the insert may include one ormore samples analytes or agent for potentially interacting with reagentsdelivered from the reagent cartridge. The samples analytes or agents arechosen for specific applications. In certain embodiments, the insertincludes a perforated membrane film strip and at least one membrane diskcoupled to a surface of the membrane film strip and aligned with anaperture on the membrane film strip. The membrane disks are each coatedwith an agent sample containing a biological and/or chemical materialsuch as a target analyte or analyte-capturing antibodies. In certainembodiments, the apertures include a central circular region and tworectangular regions open to the circular region. The rectangular regionsare configured to trap air bubbles in a fluidic flow through the assaychannel.

In another aspect, the film strip is made from a non-elastomeric plasticadhesive materials. In certain embodiments, the non-elastomer plasticmaterial includes polymethyl methacrylate, polystyrene, polycarbonateand acrylic. In certain embodiments, the membrane disks are made fromnitrocellulose, PVDF and/or nylon.

In another aspect, a heating element is coupled to the microfluidic chipfor controlling the assay temperature for enhanced assay repeatability,speed and sensitivity.

In another aspect, the invention provides a method for conductingbiological assays. After one or more sample-spotted inserts are disposedinto the appropriate assay channels, reagents from the reagent cartridgecan be flown through the assay channels via the distribution structure,thereby contacting the reaction surfaces of the inserts. Washing bufferfrom the buffer reservoir may also be flown through the assay channelsto contact the inserts in the channels. During a reaction incubationperiod or a washing period, excessive reaction reagents and/or washingbuffer in the assay channels are pumped back and forth between a shuttlereservoir and an outlet reservoir connected to each assay channel. Atthe conclusion of the assays, fluidic wastes stored in the shuttlereservoirs and the outlet reservoirs are pumped into the waste reservoirvia the distribution structure. By flowing appropriate reagents,including buffers, washing reagents, antibodies, antigens, enzymeconjugates and their substrates, the microfluidic chip can be used toperform an immunoassay or other biological assay on each membrane diskin order to detect the target analytes.

In another aspect, the shuttle reservoirs are used as reagent reservoirsfor creating individual assay conditions in each assay channel. Unlike areagent delivered from the reagent reservoir that creates uniform assayconditions in all assay channels, different reagents or reagents ofdifferent concentrations in the shuttle reservoirs may be individuallydelivered to the assay channels for performing parallel, but non-uniformbiological assays.

In another aspect, the end result of an assay is detected by colorchanges on the inserts using an automated image analysis procedure. Theprocedure involves quantitatively digitizing an array of color-spottedsamples in the assay chip and quantitatively determining the colorintensity corresponding to each pixel of a sample spot to generate anaveraged, or pixilated, value for each sample. The sample colorintensity values yield information about the biological samples oncorresponding membrane disks. A threshold value may be computed by usingnegative control samples. The threshold value, the color intensityvalues, and the various images corresponding to the sample array may bestored and archived for future reference.

In another aspect, the invention allows for porting of a microfluidicchip to a controller capable of driving the pump and valve structures onthe chip. The controller may be electronically or wirelessly connectedto a computer or a Personal Digital Assistant (PDA), such as BlackBerryor Palm Pilot, providing an interface for a user to programmably controlthe assay reactions on the chip.

The inherently small dimensions of devices achieve a portablemicrofluidic system. Combined with the programmable control directingflow of several reagents through several microchannels into severaloutlet reservoirs, this invention provides a framework for offeringportable “Point-of-Care” (POC) systems with automated assay processingthat can be run by users with little training.

In one aspect, the microfluidic chips of this invention are madeentirely from plastic materials. In one embodiment, an entiremicrofluidic chip suitable for portable immunoassay is made frompolystyrene, which results in extremely low fabrication costs. Anenabler for the use of polystyrene in such an application whilepreserving the integrity and reliability of the microfeatures disposedtherein is the use of weak solvent bonding. These aspects of thetechnology are described in U.S. patent application Ser. No. 11/242,694,incorporated by reference herein in its entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features and advantages will be more fully understood bythe following illustrative description with reference to the appendeddrawings, in which the drawings may not be drawn to scale.

FIG. 1 illustrates one embodiment of a microfluidic chip of theinvention.

FIG. 2 illustrates an alternative view of the microfluidic chip of FIG.1.

FIGS. 3 a-b illustrate a microfluidic valve used in the embodiment shownin FIG. 1.

FIGS. 4 a-4 f illustrate a microfluidic pump used in the embodimentshown in FIG. 1.

FIGS. 5 a-c illustrate an inlet valve used in the embodiment shown inFIG. 1.

FIGS. 6 a-b illustrate a cartridge and a reservoir used in theembodiment shown in FIG. 1.

FIG. 7 shows an assay chip having ducts that connect to a separatereagent chip.

FIGS. 8-10 illustrate steps for manufacturing the device of FIG. 7.

FIGS. 11 a-c illustrate an exemplary insert sized and shaped tointer-fit within the embodiment shown in FIG. 1.

FIG. 12 illustrates an embodiment of a chip in which a single drivingforce distributes a reagent to a plurality of outlet reservoirs.

FIG. 13 illustrates an embodiment of a chip in which multiple drivingforces distribute a reagent to a plurality of outlet reservoirs.

FIG. 14 illustrates an embodiment of a chip having multiple drivingforces distributing a plurality of reagents to a plurality of outletreservoirs.

FIGS. 15 a-c illustrate a method of inter-fitting the exemplary insertof FIGS. 11 a-c within a channel of the embodiment shown in FIG. 1.

FIGS. 16 a-b show the results of a microfluidic-based on-chipimmunoassay process.

FIG. 17 illustrates steps in identifying samples containing a targetanalyte.

FIG. 18 shows a complete and self-contained microfluidic systemincluding a computer, a controller and a chip.

FIG. 19 illustrates an alternate embodiment of a chip coupled to acontroller.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The invention, in various embodiments, provides microfluidic chips,systems and methods. The following detailed description refers to theaccompanying drawings. The following detailed description does not limitthe invention. Instead, the scope of the invention is at least the scopedefined by the appended claims and equivalents.

FIG. 1 illustrates a microfluidic system 1 that includes an assay chip 5and a cartridge 10 disposed on the chip 5 along a width of the chip 5.The cartridge 10 includes a plurality of reagent reservoirs 12 havingside walls that define chambers to hold fluid reagents. The chip 5includes a buffer reservoir 16 having a cylindrical sidewall to hold awashing buffer, a plurality of shuttle reservoirs 17 adapted to holdreagents during an assay operation, and a waste reservoir 18 adapted tohold used reagents and used buffer after the assay operation. The chip 5also includes a plurality of inlet valves 14 positioned to align withthe various reservoirs. The inlet valves 14 serve to control fluid flowsbetween the reservoirs and respective microchannels in the chip 5.

As illustrated in FIG. 2, the chip 5 includes a plurality of inletchannels 20, a distribution valve 25, an inlet 30, a waste channel 38, aplurality of reagent and or buffer outlet channels 35, assay channels40, fluid pumps 44, and outlet reservoirs 48. The distribution valve 25controls the release of fluid from the inlet channels 20 to the inlet30. The distribution valve 25 controls the release of fluid from theinlet 30 to the waste channel 38. The inlet 30 serves as an inlet tooutlet channels 35 which are in fluidic communication with the assaychannels 40. The pumps 44 pump fluid in a direction 60 towards theoutlet reservoirs 48, but can also be programmed to pump fluid generallyin the direction 62 towards the shuttle reservoirs 17 and the inlet 30.

As shown in FIG. 1, the chip 5 is generally constructed from a firstsubstrate 6, a second substrate 7, and a membrane 8 (not shown) disposedin between the two substrates 6 and 7. The membrane 8 has a thickness ofbetween about 10 μm and about 150 μm, or between about 15 μm and about75 μm. The depicted first substrate 6 and second substrate 7 each has athickness substantially larger than the thickness of the membrane 8, butin other implementations, has a thickness similar to or less than thethickness of the membrane 8. The microfluidic channels 20, 25, 38, and40 may be of any suitable dimension, but in certain embodiments havecross-sectional dimensions of between about 1 μm and about 500 μm, orbetween about 1 μm and about 50 μm.

In certain embodiments, the first substrate 6, the second substrate 7,and the membrane 8 are all made of plastic. Exemplary materials includenon-elastomeric polymers, such as polymethyl methacrylate, polystyrene,polycarbonate, and acrylic. These materials are beneficial at least inpart because they are reasonably rigid, which is suitable for the firstsubstrate 6 and the second substrate 7. Moreover, these materials can bedeformable when used in thin layers, which is suitable for the membrane8 which may deflect towards and away from the first 6 and second 7substrates.

The system 1 provides automated “many-to-many” reagent dispensing andprocessing. By selectively operating inlet valves 14, distribution valve25 and fluid pumps 44, various combinations of fluid flow patterns amongreagent reservoirs 12, buffer reservoir 16, waste reservoir 18, shuttlereservoirs 17 and outlet reservoirs 48 can be achieved. In particular,the distribution valve 25 may be constructed in accordance with thevalve structure described with respect to FIGS. 3 a-b. FIGS. 3 a-b showa three-layer active planar valve structure 399, which may be formedusing acetonitrile assisted bonding. The valve structure 399 includes afirst substrate 300 having interdisposed microchannels 301 and 303. Amembrane layer 304 is selectively bonded to the first substrate 300 inareas 306, thus creating a diaphragm structure 308. A second substrate302 is bonded to the membrane 304. The second substrate includes a drivechamber 310.

The channel pumps 44 of FIG. 1 may be constructed in accordance with thepump structure described with respect to FIG. 4 a-f. A microfluidic pumpgenerally refers to any structure or group of structures capable ofapplying positive and/or negative pressure to a fluid and/orfacilitating the flow of fluid in one or more desired directions. Thedepicted micro-diaphragm pump 400 generally includes three valves: aninlet valve 402, a drive valve 404 and an outlet valve 406,interconnected by portions 418 b and 418 c of microchannel 418. Inoperation, the pump 400 pumps fluid through the microfluidic channel 418by cycling through six states that are activated sequentially to producea peristaltic-like pumping effect. Even though FIG. 4 depicts threevalve structures 402, 404 and 406 that make up the pump 400, other pumpembodiments may contain four or more valve structures.

More particularly, in FIG. 4A, the inlet valve 402 opens and draws fluidfrom an inlet portion 418 a of the microfluidic channel 418 into volume425 between the membrane 408 and the second substrate 432. In FIG. 4B,the drive valve 404 opens and draws more fluid into the pump system. InFIG. 4C, the inlet valve 402 closes. In FIG. 4D, the outlet valve 406opens. In FIG. 4E, the drive valve 404 closes, and thereby forces fluidthrough the outlet valve 406 and into an outlet portion 418 d of themicrofluidic channel. In FIG. 4F, the outlet valve 406 then closes.These six states complete one pump cycle, displacing a volume of fluidthrough the pump 400.

The pump 400 is bidirectional. If the cycle is reversed, portion 418 dis an inlet portion of the microfluidic channel 418, portion 418 a is anoutlet portion of the microfluidic channel 418, and fluid flows fromportion 418 d to portion 418 a.

The valve structures 402, 404, and 408 are independently actuatable, inthat any one of the valve structures can be actuated with little orsubstantially no effect on the state of the other valve structures.Those skilled in the art will recognize that alternate sequences ofstates may produce a pumping effect, and that other pumps can also beused with this invention.

FIGS. 5 a-b illustrate an exemplary inlet valve structure 14 of FIG. 1.The valve 14 includes a first substrate 508 with a drive chamber 510fabricated therein, a second substrate 515 and a membrane 520. Areservoir may be disposed above the second substrate 515 and alignedwith reservoir port 540 to provide a source of fluid for porting intochannel 545. The reservoirs will be discussed in detail with respect toFIGS. 6 a-b. FIG. 5 c illustrates an exemplary structure including aplurality of inlet valves 14 of FIG. 1 connected in series.

Various embodiments and alternatives may be applied to the pump andvalve structures of this invention. In particular, three or more valvessimilar to the valve structure 565 in FIG. 5 c may be connected inseries by microchannels to form a pump that operates with aperistaltic-like mechanism, such as the pumps 44 of FIG. 1. Otherarrangements of valve structures interconnected by microchannels canalso form generic pumping configurations.

As described above with respect to FIGS. 5 a-b, a reservoir may bedisposed above the second substrate 515 and aligned with reservoir port540 to provide a source of fluid for porting into channel 545. This isshown in more detail in FIGS. 6 a-b. FIG. 6 a shows a cartridge 610 witha top side 602 and a bottom side 604 having a reagent reservoir 612formed thereon. In particular, the cartridge 610 is provided with itstop side 602 and bottom side 604 both sealed by suitable adhesivematerials. In the current embodiment, the top adhesive material 605 is asealing tape, and the bottom sealing material (not shown) may also be asealing tape. Other suitable adhesive materials may also be used.

FIG. 6 a depicts the cartridge 610 having only the reagent reservoirs612 disposed thereon, although various other cartridge configurationsare possible. In one exemplary arrangement, a cartridge includes abuffer reservoir 616, a waste reservoir 618 and a plurality of shuttlereservoirs 617 in addition to the reagent reservoirs 612. In certainimplementations, a cartridge includes the reagent 612 and buffer 616reservoirs. The shuttle 617 and waste 618 reservoirs may be integrallyconstructed onto the chip 615 or provided on a separate cartridge. Incertain implementations, three separate cartridges are providedrespectively including the shuttle reservoirs 617, the reagentreservoirs 612, and the buffer 616 and waste 618 reservoirs. In certainimplementations, a cartridge has only the shuttle reservoirs 617 fordistributing different reagents to assay channels 630-635.

FIGS. 7-10 illustrate an alternate method for coupling multiplereservoirs to an assay chip. FIG. 7 shows an assay chip 705, a reagentchip 710, and a ducting chip 715. The reagent chip 710 includes areagent cartridge 720 and a reagent loading chip 725. The ducting chip715 serves to provide bi-directional fluid flows between the reagentchip 710 and the assay chip 705. In particular, the reagent chip 710allows several reagent reservoirs 735-739 to dispense reagents intoreservoir 740 before being ported to the assay chip 705 through theducting chip 715. In certain arrangements, one of the reagent reservoirs735-739 may be a buffer reservoir for storing a buffer solution. Incertain arrangements, one of the reservoirs 735-739 may be a wastereservoir for storing used reagents after an assay.

The ducting chip 715 is rigid enough to provide the necessary structuralsupport to duct the assay chip 705 to the reagent chip 710. However, theducting chip 715 is deformable such that reagent chip 710 and assay chip705 need not be exactly aligned along a vertical axis 750 when they areattached by the ducting chip 715.

More specifically, according to FIG. 8 a, the ducting chip 800 includesa cover layer 805 for being generally disposed over a portion of thechannels 730, as shown in FIG. 7. The ducting chip further includes afirst support layer 810, a channel layer 815, and a second support layer820. Layers 805 and 810 are provided with apertures 825 that are alignedto allow fluid to flow from channels 830 in a downward 832 direction.The channel layer includes a plurality of inter-disposed channels 830.The first support layer 810, the channel layer 815, and the secondsupport layer 820 include apertures 845 that are substantially alignedto allow fluid to flow in a downward 832 direction from a reservoir 840.An adhesive O-ring 835 adheres the reservoir 840 to the second supportlayer 820. The layers may be adjoined with the lamination methodsdescribed herein. FIG. 8 b shows the ducting chip 800 of FIG. 8 a afterassembly.

In FIG. 9 a, the reagent loading chip 925 includes a bottom substratelayer 905 with drive chambers 907, a membrane layer 910, and a topsubstrate layer 915 with microchannels etched therein. The layers may beattached with suitable lamination methods described herein. FIG. 9 bshows a top view of the reagent loading chip 925.

FIG. 10 illustrates an exploded view of the full structure including theducting chip 1015, the reagent loading chip 1025, the reagent cartridge1020, and the assay chip 1005. In particular, FIG. 10 shows the reagentcartridge 1020 being laminated to the reagent loading chip 1025, theducting chip 1015 being coupled to the reagent loading chip 1025, andthe assay chip 1005 being attached to the ducting chip 1015.

Various alternative arrangements may be applied to the microfluidicsystems 1 and 1000 of FIGS. 1 and 10, respectively. For example, insteadof enclosed assay channels 40 as shown in FIG. 1, a plurality of voidregions 1060-1065, as shown in FIG. 10, may be disposed in therespective assay channels. These void regions 1060-1065 may be open to atop surface of the chip 1005. A cover adhesive layer may be disposedover each channel void region 1060-1065.

In another aspect, a temperature-modulating device, such as a heater ora cooler, may be coupled to the microfluidic systems 1 and 1000 toregulate the temperature of the fluids in the systems for providing anoptimal environment wherein on-chip biological and/or chemical reactionsmay occur. In FIG. 1, there are six reagent reservoirs 12, six shuttlereservoirs 17, six outlet reservoirs 48, one waste reservoir 18 and onebuffer reservoir 16. In FIG. 10, there are six reagent reservoirs1035-1039, any of which may be a buffer or waste reservoir. Howevervarious other combinations of reagent, shuttle, outlet, waste and bufferreservoirs are possible.

The assay channels may be provided with biological or chemical materialsthat react with reagents introduced into the microfluidic system. Inparticular, inserts are provided with chemical and/or biological agentsfor insertion into the microchannels for the purpose of reacting withthe reagents. Exemplary inserts are shown in FIGS. 11 a-b. In certainexamples, the insert is a flexible plastic strip with an adhesivecoating on one side. In certain examples, the insert is a thinpolystyrene strip. In certain examples, the insert has a thickness ofbetween about 50 microns to about 500 microns in thickness, a width ofbetween about 1 mm to about 5 mm, and a length of between about 5 mm toabout 100 mm. In certain instances, the assay channels are configuredaccordingly in order to accommodate the inserts disposed therein.

As mentioned above, an insert may be provided with chemical and/orbiological agents. In one exemplary implementation, an insert includes amembrane 1104 having adhesive disposed on its surface and membrane disks1110 adhered to the membrane 1104, wherein the membrane disks 1110 areprovided with chemical and/or biological agents. The membrane 1104 isfurther provided with apertures 1115 over which the membrane disks 1110lie. The apertures 1115 may be included in a perforated cover strip 1105adhering to the membrane 1104. The apertures serve to allow fluidcontact between the bottom side of the membrane disks 1110 and a fluidflow through channel 1130 wherein the insert 1107 is disposed. In oneexample of an insert as shown in FIG. 11 a, the apertures 1115 arecircular. In one example as shown in FIG. 11 b, the apertures 1115 eachincludes a central circular region 1120 with two opposing rectangularregions 1122 open to the circular region 1120. The rectangular regions122 are oriented on the insert 1107 in a direction 1132 aligned with adirection of fluid flow when the insert 1107 is disposed in the assaychannel 1130. This feature enables the insert 1107 to trap air bubblesin the fluid. The membrane disks 1110 are preferred to be circular,although other shapes are possible. The apertures 1115 are shaped andsized to provide structural support for the membrane disks 1110. For thecase of circular disks and circular apertures as illustrated in FIG. 11a, the disks 1110 are preferred to have a diameter of between about 1 mmand about 5 mm, and the apertures 1115 are preferred to have a diameterthat is between about 5% and about 10% less than the diameter of thedisks 1110. For the case of oval-shaped disks and apertures shaped asthose in FIG. 1 b, a diameter of the central circular regions 1120 ofthe apertures 115 may be between about 5% and about 10% less than amajor diameter of the membrane disks 1110. A width 1124 of therectangular regions 1122 may be between about 5% to about 10% less thanthe diameter of the central circular regions 1120.

The membrane disks 1110 may be made of a porous material such asnitrocellulose. The porosity of the membrane disks 1110 may besufficiently large to allow fluid and salt passing through but smallenough to interact with macromolecules, viruses or bacteria in thefluid. The membrane disks 1110 may be made of nitrocellulose, PVDFand/or nylon, which are suitable materials for use in amicrofluidic-based dot-chip process as will be described below. Themembrane disks 1110 and the apertures 1115 may be formed by, forexample, a die cut or laser cut. The operations of various components ofthe microfluidic system 1 of FIG. 1 will be described below. Byselectively operating the inlet valves 14, distribution valve 25, andchannel pumps 44, various combinations of fluid flow patterns might beachieved. In particular, one or more reagents stored in reagentreservoirs 12 and/or washing buffer in buffer reservoir 16 may beselectively dispensed into assay channels 40 at appropriate rates,amounts and temperatures, incubated in the channels 40 and disposedthrough waste reservoir 18 via outlet reservoirs 48 and shuttlereservoirs 17. Exemplary application of these operations will bediscussed herein.

FIGS. 3 a-b illustrate one method for operating the distribution valve25 of FIG. 1. In particular, a positive upward pressure is applied tothe diaphragm 308 via the drive chamber 310, the membrane 308 is pushedaway against the valve seat 312 between the two microfeatures 301 and303, effectively preventing any transfer of fluid between them.Alternatively, if a negative downward pressure is applied to the drivechamber 310, the membrane 308 is pulled away from the valve seat 312 andthe fluid is free to communicate between the microfeatures 301 and 303via void region 314. Pressure may be applied through the drive chamber310 pneumatically or by physically contacting the membrane through thedrive chamber 310.

FIGS. 4 a-f illustrate one method for pumping fluid through the pumpstructure 44 of FIG. 1. The method comprises cycling the pump structurethough six states that are activated sequentially to produce a pumpingeffect. In FIG. 4 a, the inlet valve 402 is opened and fluid is drawnfrom inlet microchannel 412 into the volume 402 a between the membrane408 and the first substrate 410. In FIG. 4 b, the drive valve 404 isopened, drawing more fluid into the pump system. In FIG. 4 c, the inletvalve 402 is closed. In FIG. 4 d, the outlet valve 406 is opened. InFIG. 4 e, the drive valve 404 is closed, forcing fluid out through theoutlet valve 406 into outlet microchannel 418. The outlet valve 406 isthen closed. These six states complete one pump cycle, displacing avolume of fluid through the pump. The pump is bi-directional. If thecycle is reversed, microchannel 418 serves as an inlet microchannel,microchannel 412 serves as an outlet microchannel, and fluid may bedrawn from inlet microchannel 418 to outlet microchannel 412. Thoseskilled in the art will recognize that alternate sequences of states mayproduce other pumping effects.

FIGS. 5 a-b illustrate one method for operating the inlet valves 14 ofFIG. 1. In particular, a positive pneumatic force 525 is applied throughdrive chamber 510, forcing the valve 500 to be in a closed positionwherein there is no fluidic communication between inlet channel 545 andreservoir port 540. Upon application of a negative pneumatic force 530through drive chamber 510, the valve 500 is in an open position whereinreservoir port 540 is in fluidic communication with inlet channel 545.

FIG. 5 c illustrates the operation of a plurality of inlet valves beingconnected in series. As depicted, communication between inlet valves 550and 557 may be controlled by actuating a valve structure 565 connectedto the inlet valves. In particular, a positive pneumatic force 570 maybe applied through the drive chamber 586 disposed in the bottomsubstrate 593. This force will push the membrane 588 into conformalcontact with a region 590 of the top substrate 592. In this case, thevalve is in a closed position with substantially no fluidiccommunication between adjoining microchannels 572 and 573. A negativepneumatic force 575 applied through the drive chamber 586 will pull themembrane 588 away from the top substrate 592, such that the membrane 588forms a cavity towards the drive chamber 586 into the region 587. Inthis case, the valve is in an open position in which adjoiningmicrochannels 572 and 573 are in fluidic communication.

With reference to FIG. 6 b, to couple the cartridge 610 to the assaychip 615, a user turns the cartridge 610 such that its bottom side 604is facing up, removes the bottom sealing backing, aligns the cartridge610 to the assay chip 615 such that the reagent reservoirs 612 arealigned with respective valves 614, and then presses the assay chip 615against the cartridge 610. When the reagent cartridge is held togetherwith the assay chip 615, reagent 620 within the respective reagentreservoir 612 is maintained within the reagent reservoir 612 by ahydrophobic property of the surface of aperture 624. Subsequent the chipassembly may be placed on a controller (not shown) and the cover sealingtape is removed to release the reagent 610 onto the assay chip 615 byactuating corresponding valves and pumps described below.

FIGS. 12-14 illustrate various embodiments for distributing fluidsthrough the chip 1 of FIG. 1 by actuating the pump and valve structuresdescribed above. FIG. 12 illustrates a single driving force fordistributing a reagent from a reagent reservoir 1205 a among a pluralityof microchannels 1220-1223 on a chip 1200. The single driving force isproduced by an inlet valve 1215 a and a drive diaphragm 1224 located inbetween the area of an inlet valve 1215 a and an outlet valve 1225.These three valves may operate according to the peristaltic-like pumpingmechanism described above with respect to FIG. 4 to transport fluidcontents of reservoir 1205 a among the outlet channels 1210-1213.Similarly, reagent contents of reservoirs 1205 b-d may be delivered tooutlet channels 1210-1213 via pumping action produced by respective onesof inlet valves 1215 b-d, drive diaphragm 1224 and outlet valve 1225.This results in a “many-to-many” functionality wherein several reagentsare being distributed to several outlet reservoirs.

However, the flow resistances of outlet channels 1210-1213 impact thefluid flow rate on assay channels 1220-1223. In particular, the flowrate in each channel of an assay chip is inversely proportional to theflow resistance of that channel. The outlet channels 1210-1213 may befabricated to have different flow resistances if an application callsfor different channels to have different respective flow rates. However,the sensitivity of flow rates to channel resistance is a detriment toreagent processing if the varying resistances among channels isunintentional. In particular, air bubbles formed during assay may resultin varying flow resistances which cause an uneven distribution ofreagent across the assay channels 1220-1223.

FIG. 13 illustrates an embodiment of the chip 1 in FIG. 1 that overcomesthe variation in flow rates resulting from varying channel flowresistances. Each assay channel 1310-1315 and each outlet channel1360-1365 are associated with a respective fluid pump 1320-1325. Theamount of fluid delivered to the channel by each of the pumps 1320-1325is relatively unaffected by variations in flow resistance among theassay channels 1310-1315 when the flow resistance is substantiallysmaller than the pneumatic driving force used to operate the fluid pumps1320-1325. The channel-to-channel flow rate variation is dominated bythe characteristics of pumps 1320-1325 rather than channel flowresistances. FIG. 13 illustrates a reagent from reagent reservoir 1350being distributed (see arrows) among outlet channels 1360-1365 viadistribution valve 1352. In certain embodiments, a plurality of reagentsfrom their respective reagent reservoirs 1350-1355 are delivered to thedistribution valve 1352 wherein the reagents may be mixed to create areagent mixture. In certain embodiments, the reagent or reagent mixturemay be further distributed to selected assay channels 1310-1315, outletreservoirs 1330-1335, and/or shuttle reservoirs 1340-1345.

FIG. 14 illustrates additional fluid distribution patterns of themicrofluidic system shown in FIG. 1. In particular, each shuttlereservoir 1440-1445, assay channel 1410-1415 and outlet channel1460-1462 are connected in series to form a fluid pump 1420-1425,wherein each fluid pump 1420-1425 provides bi-directional fluid flow toand from the respective micro-features. In one implementation, fluidpumps 1420-1425 provides bi-directional fluid flow between shuttlereservoirs 1440-1445 and outlet reservoirs 1430-1435 interconnected bythe respective assay channels 1410-1415. In one implementation, areagent in outlet reservoir 1432 is delivered through outlet channel1461 and distribution valve 1462 to waste reservoir 1464. In oneimplementation, a reagent in shuttle reservoir 1443 is delivered towaste reservoir 1464 via outlet channel 1461 and distribution valve1462. In one embodiment, different reagents or reagents of differentconcentrations may be introduced to the assay channels 1410-1415 fromthe corresponding shuttle reservoirs 1440-1445. Introducing reagentsfrom shuttle reservoirs permits variability in assay channel conditionsthrough tailored reagent delivery.

As will be discussed with respect to FIGS. 19-20, the pumps and valvesof FIG. 1 may be selectively and programmably actuated. In particular,by selectively actuating certain inlet valves 14, a user may releaseselected reagents stored in selected reagent reservoirs 12 and/orwashing buffer stored in buffer reservoir 16. By selectively actuatingchannel pumps 44, a user may store these fluids in selected shuttlereservoirs 17 and outlet reservoirs 48, release these fluids stored inthe selected shuttle reservoirs 17 and outlet reservoirs 48, and storethese fluids in waste reservoir 18. Thus a user is able to perform anydesired combination of incubation/mixing/reacting/aspiration of thefluids in the reagent 12 and buffer 16 reservoirs.

The microfluidic system 1000 of FIG. 10 separates the assayfunctionality of the invention from the reagent delivery functionality.In situations where a particular assay needs to be performed repeatedly,it may be more inconvenient to use a larger cartridge repeatedly thanseveral smaller ones. In one example, the microfluidic system 1000 maybe used to run a number of identical assays in parallel. Thus thereagent reservoirs 1035-1039 are provided with enough reagents to runseveral assays, and the reagent chip 1010 supplies reagent to severalchips as their respective assays are being performed. In anotherexample, ducting chip 1015 may be used to duct used reagents from assaychip 1005 into reservoir 1040 on reagent chip 1005. The used reagent inreservoir 1040 is then ported to waste reservoir 1035 for disposal.Waste reservoir 1035 may be utilized to store used reagents from one ormore assay chips.

The microfluidic system 1000 operates by flowing fluids from reagentreservoirs 1035-1039 into reservoir 1040. A fluid may be delivered fromreservoir 1037 to reservoir 1040 via valve 1041 much like the processshown in FIG. 5 c according to which a fluid from valve 550 is deliveredtoward valve 555 via valve 565. More specifically, actuating valve 1050delivers fluid into channel 1072, actuating valve 1041 delivers fluidinto channel 1073, and actuating valve 1055 delivers fluid intoreservoir 1040. In another aspect, a fluid flows from reservoir 1040into a reagent reservoir 1036 by a similar mechanism as that illustratedin FIG. 5 c. For example, with valves 1055, 1041 and 1062 all in openstates, actuating valve 1055 pushes fluid into channel 1073, actuatingvalve 1041 pushes fluid into channel 1064, and actuating valve 1062pushes fluid into reservoir 1035.

As illustrated in FIG. 11 c, to conduct an assay using a microfluidicsystem 1100 of the invention, the insert 1107 is first deposited into anassay channel 1130 through an opening of the outlet reservoir 1134 thatis located at the end of the assay channel 1130 and has a widthsubstantially the same as the width of the assay channel 1130. Theinsert 1107 is slid into the channel 1130 until it spans a length 1136of the channel. In certain embodiments as illustrated according to FIG.7, the insert is inserted into the assay channel 760 through channelvoid 730. In particular, the channel void 730 is provided with an opentop in which the insert is disposed. The insert is slid into the channel730 until it spans a length 762 of the covered portion of the channel760. After insertion, an adhesive cover may be placed over the channelvoid region 730 to form shuttle reservoirs at the end of the assaychannel 760.

FIG. 15 a illustrates the insertion of an insert 1507, and inparticular, shows an exemplary channel structure that facilitates theuse of the insert 1507. The channel 1520, as shown from across-sectional view in FIG. 15 b, is a stepped channel including a widebottom portion 1522 and a narrow top portion 1524. The insert 2017 isinserted into the stepped channel 1520 such that it generally overliesmembrane 1510, as shown in FIG. 15 c. More specifically, FIG. 15 c showsthe insert 1507 having an aperture 1515 and a membrane disk 1525. Theinsert 1507 is situated in the channel 1520 such that the top surface ofthe membrane disk 1525 does not contact a top surface 1517 of thechannel 1520, allowing for fluid in channel 1520 to flow around andcontact the membrane disk 1525.

In one aspect, the insert is used to perform an assay similar inprinciple and function to a dot-ELISA method. The dot-ELISA is a method,known in the art, for detecting the presence of a target analyte withinsamples. Drawbacks of the conventional dot-ELISA process includedifficulties with standardization. Many of the steps are often performedby hand in Petri dishes and the specification of these procedures isvague. Additionally, sample locations are hardly controllable. Whensample is spotted on a membrane surface, the hydrophilicity of thematerial may lead to rapid sample spreading and diffusion. Larger sampleamounts result in larger spotted areas. Moreover, since detectionsensitivity is related to analyte density per unit area, this diffusionmeans that larger sample amounts do not necessarily result in lowerdetection limitation. The present invention employs a similar assayprocessing, but allows for standardized and more efficient handling,treatment, and analysis. In particular, samples are applied to amembrane disk 1110 as shown in FIGS. 11 a-b. The samples are air dried,and then the insert 1105 is disposed in an assay channel of amicrofluidic chip, similar to that of FIG. 1.

With reference to FIG. 1, the operation of the microfluidic chip 1 inperforming assays will be discussed. Various reagents are stored inreagent reservoirs 12 for conducting on-chip immunoassay. The reagentsinclude fluids that will be employed in a dot-ELISA assay. Morespecifically, various reservoirs may include one or more of bufferwashing buffer, antibody, antibody with conjugated enzyme, and enzymesubstrate. In some cases, a buffer reservoir 16 may be used to store awashing buffer. The buffer reservoir 16 may feature a substantiallylarger void volume than the individual reagent reservoir 12. Thereagents are released from their respective reservoirs 12 by activatingrespective inlet valves 14 and then distributing the reagents throughoutthe assay channels 40 using the activation of distribution valve 25 andchannel pumps 44. The washing buffer in buffer reservoir 16 may also bereleased into the assay channels 40 in a similar manner. The order andtiming of release of the reagents and buffer from their respectivereservoirs will correspond to the steps of the assay method used. By wayof example, the reagents may correspond to the reagents described abovewith respect to the immunoassay process, and are released in accordancewith the order and timing of the steps mentioned above. The releasedreagents flow through the assay channels 40 and contact the inserts 70therein. With respect to FIG. 15 c, a fluid flowing through the narrowportion 1524 of the stepped channel 1520 contacts and reacts with agentson the membrane disks 1525. Apertures 1515 provide for the possibilityof additional fluid contact along a bottom side of the membrane disks1525.

As mentioned above, the channels may be provided with materials withwhich the fluid reagents react, i.e., reagents may flow through assaychannels with membrane disks disposed therein, thereby causing theoccurrence of interactions between the reagents and the analysts on themembrane disks. It may be desirable to allow dynamic flow conditions orlonger incubation times for the reactions via multiple passes of thereaction reagent through channels. This is achieved in part by thebidirectional pumping functionality of this invention. In particular,with reference to FIG. 1, the bidirectional channel pumps 44 are used torepeatedly shuttle a reagent back and forth between the shuttlereservoirs 17 and outlet reservoirs 48 along respective assay channels40. This cycling action provides multiple passes for much greaterefficiency at longer reaction time. The outlet reservoirs 48 and shuttlereservoirs 17 are directly vented to the atmosphere, thereby allowingrelease of air from the channels 40 during the pumping cycles. Incertain examples, the void volume of each shuttle reservoir 17 and eachoutlet reservoir 48 are substantially larger than the void volume ofeach assay channel 40 so that reagents in the channels 40 may be storedin the reservoirs during the back and forth pumping action. After theassay operation, used reagents are then transported to the wastereservoir 18 for disposal. In one example, the void volume of wastereservoir 18 is substantially larger than the void volume of the bufferreservoir 16 for storing all used reagents and washing buffer after anassay operation. After the inserts are treated with different reagents,the color of the membrane disks may be observed for the presence of atarget analyte in the samples.

The systems described herein bring several new assay advantages to aconventional dot-ELISA format assay. In particular, with reference toFIG. 11, the hydrophobic nature of the insert 1107 along with theinherent surface tension of the liquid sample allows a user to apply alarger amount of sample to a membrane disk 1110 without diffusion orspreading of the sample to other disks 1110 nearby. In oneimplementation, sample spotting onto the insert 1107 is accomplished byplacing the insert 1107 on an absorbent backing material such as achromatograph paper with membrane disk surface touching the paper. Thecombination of the water-absorbent ability of the backing material andthe sample-retaining ability of the insert 1107 give rise to rapidsample absorption and concentration effects during spotting.Furthermore, the sample droplet diffusion area is substantially definedby the area of the membrane disk 1110. This results in severaladvantages, such as after a larger amount of sample has dried on themembrane disk, a higher density of sample within the area defined by themembrane disk 1110 is achieved. In addition, since there is less risk ofdiffusion and contamination of sample material between differentmembrane disks 1110, the membrane disks 1110 may be placed closertogether than the sample spots 2210 would be placed on the monolithicmembrane 2205 as shown in FIG. 22, thus resulting in improved spaceefficiency for on-chip processing and potential reagent savings.Moreover, placing the membrane disks 1110 at predefined and well knownlocations along the insert 1107, with embedded barcodes or otheridentifiers on-chip, facilitates the use of the assay chip in automateddata processing and image analysis methods that make data archiving foron-chip immunoassay results much more useful.

FIG. 16 a illustrates a plurality of inserts 1705 in channels after anassay has been performed. As shown, certain membrane disks 1710 a havebeen colored as positive results by an enzyme-substrate reaction,indicating the presence of a target analyte in a sample disposed on thecorresponding membrane disk. Other membrane disks 1710 b aresubstantially not colored, indicating no target analyte in a sampledisposed on the corresponding membrane disk. In a preferred arrangement,each insert 1705 includes eight membrane disks 1710. Each chip mayinclude six or more assay channels, and therefore at least 48 samplesmay be assayed simultaneously.

In one implementation, an image analysis method is provided for theautomated processing of on-chip immunoassay results. In particular, amicrofluidic chip may be scanned utilizing, for example, a photo scanneror a digital camera to capture one or more colored images of the insertsafter an assay operation. FIG. 16 a provides an exemplary image of an8×6 sample-spotted array. In one embodiment, the scanned images may bestored in a handheld device for further off-line manipulation or sent toa remote computer for off-line image analysis. Image analysis softwaremay then be used to analyze the color intensities of the membrane disksfrom the captured color images. The intensity of each membrane disk 1710is subsequently digitized into pixels with a numerical value assigned toeach pixel. By averaging the numerical values of the pixels for eachmembrane disk, one may systematically determine a color intensity valuecorresponding to the membrane disk 1710. FIG. 16 b illustrates anexemplary array of color intensity values 1716 corresponding to themembrane disk array shown in FIG. 16 a.

In one embodiment, each membrane disk 1710 in a sample array is uniquelyidentifiable by a combination of a barcode embedded in the chip and aset of coordinates specifying the channel and insert positions at whicha membrane disk is located. For example, as shown in FIG. 16 a, amembrane disk 1710 c on the upper-left corner of a chip that isbar-coded as CHIP-0001 may be labeled as CHIP-0001-A1, where A1indicates a combination of the column 1712 and row 1714 positions wherethe disk 1710 c lies. Hence, placing the membrane disks 1710 atpredefined locations on a bar-coded chip enables their correspondingcolor intensity values 1716 to be easily archived in a database forfuture reference.

In one example, a protocol is provided for interpreting a colorintensity value 1716 for identifying the presence of a target analyte ina sample disposed on the corresponding membrane disk 1710. According tothe protocol, a threshold value is computed using negative control diskssuch that a color intensity value 1716 is interpreted as having apositive result for target analyte if the color intensity value is abovethe threshold value. FIG. 17 provides an illustration for determiningthe presence of a target analyte in eight exemplary samples. Thesesamples are disposed on membrane disks 1814 and correspond to computedcolor intensity values 1812. The threshold value 1810 in this particularembodiment is 26.8 by arithmetically averaging C1, F1, B2, E2, H2, C3,F3, B5, E5 and H5 as shown in FIG. 16 b. As shown, the membrane disks1814 in positions A, B, D, E, G, H are identified as having coated withthe target analyte-containing solution. This automated identificationprocedure reduces human reading errors, especially when interpretingsamples, such as that in position F, where the corresponding colorintensity 1814 a is fairly close to the threshold value 1810.

The samples and target analytes for the assay may be any samples andtargets suitable for use with immunoassay processes. The samples mayinclude control samples and experimental samples. Experimental samplesare generally taken from a subject with a condition of interest, andcontrol samples generally mimic the subject but exclude the analyst ofinterest. Typically, experimental samples are taken from a potentiallydiseased patient. A subject may be, for example, a human, animal orplant.

FIG. 18 shows a complete system including an assay chip 1905, acartridge 1910, a controller 1915, and a computer 1920. The controller1915 allows for automated control of the various pump and valvestructures of the chip 1905. In particular, the chip 1905 includespneumatic drivers 1920 (not shown) positioned to be substantiallyaligned with the pump and valve structures of the chip 1905. Positive ornegative pneumatic pressure is applied via the drivers 1920 inaccordance with input signals provided through input wires 1925.

The computer 1920 may provide a user interface for controlling thecontroller 1915. A user may provide inputs specifying requirements on aparticular assay run using a graphical user input provided by thecomputer 1920. The computer is electrically connected to the controller1915 and provides signals to the controller 1915 so it acts inaccordance with the user inputs.

FIG. 19 illustrates an embodiment with an assay chip 2005 ducted to aseparate reagent chip 2010 on a programmable controller 2015. Thecontroller 2015 includes a group of pneumatic solenoid valves. Each ofthe pneumatic signals from the solenoid valves is routed through thechip to one or a series of microfluidic valves on a specific chiplayout. For example, in one embodiment there is an individual solenoidvalve connected to each of the corresponding reagent reservoirs 12 ofFIG. 1, but all six of the channel pumps 44 are connected in parallel toa set of four solenoid valves so they may act together. There is asolenoid drive board in the controller 2015 that takes the signals fromthe computer and turns on the appropriate solenoid valve to actuate therequired microfluidic valve. An electrical signal from the computer willcause a solenoid valve to switch from a normally pressurized state to avacuum state. This opens the attached microfluidic valve. If a specificsequence of solenoid valve actuations is to be run repeatedly, thecomputer connection to the controller is not necessary. Themicroprocessor on the control board includes a memory which may storethe sequence and thus an assay may be run independently of externalcomputer control.

As mentioned above with respect to FIG. 1, the microfluidic chip of theinvention generally includes a top substrate 7, a bottom substrate 6,and a membrane 8 disposed therebetween. The microfeatures (e.g., pumps,valves, or reservoirs) are fabricated in one or more of the topsubstrate 7, the bottom substrate 6, and the membrane 8. In certainmethods of fabrication, the top substrate 7 and the membrane 8 arelaminated together, and similarly the membrane 8 and the bottomsubstrate 6 are laminated together. While any lamination method known inthe art may be used, in one aspect of the invention these layers arelaminated by: 1) using a weak solvent bonding agent, and 2) laminatingthe layers under mild conditions, such as under low heat or lowpressure. This is beneficial at least in part because this laminationmethod reduces or eliminates damage to the microfeatures during thelamination process. More particularly, in an exemplary use, the weaksolvent bonding agent is applied to one or both surfaces to be adhered,and then mild pressure (e.g., from moderate heat or moderate physicalpressure pressing the surfaces together) adheres the surfaces.

According to an aspect, the weak solvent bonding agent may be chemicallydefined as:

where, R1=H, OH or R, where R=alkyl, or is absent, R2=H, OH or R, whereR=alkyl, or is absent, and R2=H, OH or R, where R=alkyl, or is absent.

Alternatively, the weak solvent may have a chemical formula of:

where R1=H, OH or R, where R=alkyl, or is absent, and R2=H, OH or R,where R=alkyl, or is absent.

Alternatively, the weak solvent may have a chemical formula of:

where R1=H, OH or R, where R=alkyl, or is absent.

In a particular aspect, the weak solvent bonding agent is acetonitrile.Acetonitrile is a versatile solvent that is widely used in analyticalchemistry and other applications. It is 100% miscible with water andexhibits excellent optical properties. The ability of acetonitrile tohave little or no effect on polymeric surfaces under ambient conditionsbut adhere, the surfaces under moderate pressure makes it highlysuitable for laminating polymeric materials such as polystyrene,polycarbonate, acrylic and other linear polymers. For example,microstructures disposed on a polystyrene substrate that was treatedwith acetonitrile at room temperature for at least several minutes didnot exhibit any noticeable feature damage.

While some materials may be more susceptible to damage from acetonytrilethan polystyrene, this increased susceptibility may be controlled byapplying the acetonitrile at a lower temperature or, alternatively, byusing a combination of acetonitrile and other inert solvents.

An additional benefit of acetonitrile-based lamination is that theprocess allows substrate alignment for structures containingmulti-component layers or fluid networks constructed utilizing both acover plate and a base plate. Unlike conventional strong solventlamination, which tends to penetrate the polymeric surface and create atacky bonding surface within seconds of solvent application,acetonitrile at room temperature may gently soften the surface. When twosurfaces with acetonitrile disposed thereon are placed in contact atlower temperature prior to applying pressure, an operator may slide thetwo surfaces against each other to adjust their alignment. Afteraligning the surfaces, the operator may then apply pressure to thesurfaces to laminate them together.

The foregoing description of the preferred embodiment of the inventionhas been presented for the purposes of illustration and description. Itis not intended to be exhaustive or to limit the invention to theprecise form disclosed. Many modifications and variations are possiblein light of the teaching herein.

1. A microfluidic device, comprising: a substrate having a plurality ofchannels disposed therein, each of the channels having an inlet end andan outlet end; at least one reagent reservoir of a type capable ofholding a material; a plurality of outlet reservoirs coupled torespective outlet ends of the plurality of channels; at least onebi-directional diaphragm pump comprising at least three non-elastomericmembrane-based valve structures; a distribution valve disposed in fluidcoupling with the at least one reagent reservoir and at least one of theinlet ends, wherein the distribution valve is adapted to controllablydirect a flow of the material from the at least one reagent reservoir toa plurality of outlet reservoirs via at least one of the channelscoupled to the distribution valve; and a cartridge adapted to detachablycouple to the substrate and having the at least one reagent reservoirdisposed thereon for porting the material into the plurality of channelsdisposed in the substrate.
 2. The device of claim 1, wherein the insertremovably inter-fits within a void volume of the at least one of thechannels, wherein a gap is provided between the reaction surface of theinsert and a surface of the void volume suitable for allowing thematerial to pass there through.
 3. The device of claim 2, wherein thevoid volume is adapted to receive the insert from an opening of at leastone reservoir fluidly coupled to the void volume, wherein a width of thevoid volume is at least about equal to a width of the insert.
 4. Thedevice of claim 2, wherein the void volume is adapted to receive theinsert from an opening of the void volume, and a lid member is providedfor removably covering the opening.
 5. The device of claim 1, whereinthe substrate includes a plurality of laminated layers having theplurality of the channels disposed in at least one of the layers.
 6. Thedevice of claim 1, wherein the material is one of a fluid material, agaseous material, a solid material that is substantially dissolved in afluid material, a slurry material, an emulsion material, and a fluidmaterial with particles suspended therein.
 7. The device of claim 1,further comprising at least one heating element for adjusting atemperature of the material.
 8. The device of claim 1, furthercomprising: at least one pump having a plurality of independentlyactuatable valve structures, and a programmable controller capable ofpneumatically actuating the valve structures for directing a materialflow from the at least one reagent reservoir to the plurality of outletreservoirs via at least one of the channels coupled to the distributionvalve.
 9. The device of claim 1, wherein the at least one regentreservoir is removably coupled to a top surface of the substrate,further wherein the least one reagent reservoir is fluidly coupled to atleast one of the plurality of channels.
 10. The device of claim 1,wherein the distribution valve is coupled to at least one bi-directionalpump to transport the material from the at least one reagent reservoirto at least one of the plurality of outlet reservoirs via an actuationof the pump.
 11. The device of claim 1, further comprising at least onereagent valve having open and closed states and fluidly couples the atleast one reagent reservoir to the plurality of outlet reservoirs viathe distribution valve.
 12. The device of claim 1, further comprising atleast one bi-directional pump associated with at least one of thechannels.
 13. The device of claim 12, wherein the bi-directional pumpincludes at least three independently actuatable valve structures beingconnected in series by respective ones of the channels.
 14. The deviceof claim 12, wherein the at least one of the channels connects a firstreservoir and a second reservoir, and the at least one bi-directionalpump is adapted to shuttle the material in a first direction towards thefirst reservoir and in a second direction towards the second reservoir.15. The device of claim 1, further comprising a plurality ofbi-directional pumps associated with respective ones of the channels,wherein the distribution valve fluidly couples to respective ones of thechannels via corresponding ones of the bi-directional pumps, and, whenin a closed state, the distribution valve substantially prevents fluidcommunication among the channels and between the at least one reagentreservoir and the channels.
 16. A microfluidic device, comprising: asubstrate having a plurality of channels disposed therein, each of thechannels having an inlet end and an outlet end; at least one reagentreservoir of a type capable of holding a material; a plurality of outletreservoirs coupled to respective outlet ends of the plurality ofchannels; at least one bi-directional diaphragm pump comprising at leastthree non-elastomeric membrane-based valve structures; a distributionvalve disposed in fluid coupling with the at least one reagent reservoirand at least one of the inlet ends, wherein the distribution valve isadapted to controllably direct a flow of the material from the at leastone reagent reservoir to a plurality of outlet reservoirs via at leastone of the channels coupled to the distribution valve; at least oneinsert configured to inter-fit within at least one of the channels andhaving a reaction surface for interacting with the material, wherein theinsert comprises a membrane.
 17. The device of claim 16, wherein theinsert further comprises at least one membrane disk disposed over anaperture of an adhesive layer of the membrane.
 18. The device of claim17, wherein the aperture is one of a circular shape and a shapecomprising a circular region and two opposing rectangular regions opento the circular region.
 19. The device of claim 17, wherein the membranedisk is applied with at least one of a chemical agent and a biologicalagent.
 20. The device of claim 17, wherein the membrane disk is madefrom one of nitrocellulose, PVDF, polystyrene, and nylon.